Manufacture of Vaccines and Compositions for the Prevention of Salmonella Infections

ABSTRACT

Disclosed are stable conjugate vaccine formulations for protection against  Salmonella typhi,  and methods of conjugation between Vi-polysaccharide of  S. typhi  to tetanus toxoid as the carrier protein, responsible for producing improved T-dependent immune response against Typhoid fever caused by  Salmonella typhi.  The methods disclosed in this invention and the resulting formulations are capable of inducing immunity against typhoid fever including in children below 2 years of age, through only a single injection to comprise a complete vaccination schedule.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.14/913,816 filed Feb. 23, 2016, which is a national phase applicationunder 35 U.S.C. § 371 of International Application No. PCT/IN2014/00530filed Aug. 19, 2014, which claims priority to Indian Patent ApplicationNo. 3750/CHE/2013 filed Aug. 24, 2013, the disclosures of which areincorporated by reference herein.

FIELD OF THE INVENTION

The invention relates to the field of bacterial vaccine characterized toraise adequate immune responses in infants, children and adults againsttyphoid fever. Particularly, the present invention relates to conjugatevaccines and processes of manufacture thereof, wherein the nativepolysaccharides of Salmonella typhi are conjugated to carrier proteinsand formulated as a prophylactic conjugate vaccine. Furthermore, thisinvention also relates to the field of combined vaccine formulations forprotection against Salmonella typhi and measles virus.

BACKGROUND OF THE INVENTION

Salmonella typhi, the causative bacterium for typhoid fever in humanbeings is a major endemic disease in Africa, Asia, and Middle East. Foodand water contaminated with S. typhi bacterium was identified as majorsource in transmission of the disease. Various studies have shown thatthe global burden of typhoid fever varies in different parts of theworld. More than 100 cases in 100,000 populations per year reported inSouth Central Asia and South-East Asia; Asia, Africa, Latin America andthe Caribbean are estimated to have medium incidence of typhoid fever,i.e., 10 to 100 cases in 100,000 populations per year; New Zealand,Australia and Europe have low to very low incidence (Crump et al.,2004). This suggests that Typhoid fever is strongly endemic in theregions of the World particularly in the developing nations andcountries with low resource settings.

Salmonella belongs to the family of Enterobacteriaceae that includes thegenera Shigella, Escherichia, and Vibrio. The genus of Salmonellacontains two different species, S. enterica and S. bongori. S. entericais further divided into six subspecies (enterica, salamae, arizonae,diarizonae, houtenae and indica) containing 2443 serovars. The agentsthat cause enteric fever are therefore Salmonella enterica subspeciesenterica serovar typhi (commonly referred to as S. enterica serovartyphi) and serovars Paratyphi A, B and C. A serovar or serotype can bedefined as a strain that has a unique surface molecule which isresponsible for the production of specific antibody. Each serotype hassubtle chemical differences in their antigenic region (Brenner et al.,2000).

Salmonella typhi has a combination of characteristics that make it aneffective pathogen. This species contains an endotoxin typical of gramnegative organisms, as well as the Vi polysaccharide antigen which isthought to increase virulence. It also produces and excretes a proteinknown as “invasin” that allows non-phagocytic cells to take up thebacterium, allowing it to live intracellularly. It is also able toinhibit the oxidative burst of leukocytes, making innate immune responseineffective. During the last decade, Salmonella species have been foundto acquire more and more antibiotic resistance. The cause appears to bethe increased and indiscriminate use of antibiotics in the treatment ofSalmonellosis of humans and animals, and the addition ofgrowth-promoting antibiotics to the food of breeding animals.Plasmid-borne antibiotic resistance is very frequent among Salmonellastrains involved in pediatric epidemics. Resistance to ampicillin,streptomycin, kanamycin, chloramphenicol, tetracycline, ceftriaxone,cefotaxine, cefoperazone and sulfonamides is commonly observedColistin-resistance has not yet been observed. Salmonella strains shouldbe systematically checked for antibiotic resistance to aid in the choiceof an efficient drug when needed and to detect any change in antibioticsusceptibility of strains (either from animal or human source). Until1972, Salmonella typhi strains had remained susceptible to antibiotics,including chloramphenicol (the antibiotic most commonly used againsttyphoid); but in 1972 a widespread epidemic in Mexico was caused by achloramphenicol resistant strain of Salmonella typhi. Otherchloramphenicol-resistant strains have since been isolated in India,Thailand and Vietnam.

Vaccination against typhoid fever caused due to Salmonella Typhi isessential for protection against these life-threatening disease due toincreasing antibiotic resistance. It is also an important protectivetool for people travelling into areas where typhoid fever is endemic. Asthe bacterium has the ability to acquire multi-drug resistance ability,antibiotics may not offer complete protection. Three types of typhoidvaccines have been made currently available for use till now: (1)Parenteral killed whole cell vaccine; (2) Oral live-attenuated vaccine;and (3) Typhoid-Vi capsular polysaccharide vaccine for parenteral use.Vaccines against typhoid fever were designed in early ages when theorganism's cellular and molecular complexity was studied clearly.Initially parenterally administered whole cell S. typhi killed byheat-phenol-inactivation method was used as a vaccine, to beadministered in two doses. Since the whole cell inactivated vaccinescontain the ‘O’ antigen (endotoxin), they tend to produce local andgeneral reactions in vaccinated individuals and these types of vaccinesrequired a booster dose for every two years. Oral live-attenuated Ty21avaccine are considered as second generation vaccines prepared withmutant S. typhi strain lacking adenylate-cyclase and AMP receptorprotein and mutants auxotrophic for p-amino benzoate and adenine. Theselive attenuated vaccines reported poor efficacy and was found to be notsuitable for administration of children's below 6 years of age.Additionally, a booster dose is also required for every 5 years.Subsequently, capsular Vi-polysaccharide of S. typhi was identified as aprotective immunogen capable for eliciting adequate immune responses inhumans and hence used as a potential vaccine candidate in routineimmunization schedule. A dose of 25 μg/0.5 mL injection of purifiedcapsular Vi-polysaccharide (ViPs) can produce maximum sero-conversioni.e. fourfold rise in antibodies. However, the limitations of theVi-polysaccharide vaccine have been reported in many clinical trialsthat native polysaccharide vaccine are incapable or do not producesecondary memory responses. This phenomenon is because of bacterialpolysaccharides are T-cell independent in nature and hence are notcapable to produce cell mediated immune responses. Therefore to overcomethe said problem, polysaccharides of S. typhi and carrier proteins werefurther conjugated to form polysaccharide-protein molecules to make itT-cell dependent antigens. There are various factors that influence thecoupling of polysaccharides and proteins which depend upon molecularweight of the ViPs and carrier proteins selected and activation of thefunctional groups. Low molecular weight polysachharides can result inefficient coupling to carrier proteins. Different carrier proteins liketetanus toxoid, diphtheria CRM 197, the B subunit of the heat-labiletoxin (LT-B) of Escherichia coli, the recombinant exoprotein A (rEPA) ofPseudomonas aeruginosa, Flagellin Fli C, and Horseshoe rab Haemocyanin(HCH) have been mostly used for conjugation.

WO1996/011709 discloses an O-acetylated oligonucleotide orpolygalactouronate pectin which is substantially identical to Vipolysachharide subunit structure conjugated to a carrier protein tetanustoxoid wherein the carrier protein being derivatized with cystamine Thisparticular patent teaches to conjugate an identical polysaccharide butnot Vi-polysaccharide to carrier protein with a different derivatizerthat is cystamine, Subsequently, WO1998/026799 discloses an isolatedlipo-polysachharide from Salmonella Paratyphi A, having removed itsLipid A through detoxification and retaining its O-acetyl contentbetween 70% to 80% and then conjugated to a carrier protein tetanustoxoid through adipic acid dihydrazide (ADH). WO2000/033882 discloses aVi-polysaccharide of the Salmonella typhi covalently bound to a proteinpseudomonas aeruginosa (Vi-rEPA) conjugate through adipic aciddihydrazide. WO2007/039917 discloses an exogenous antigen of Salmonellatyphi which is covalently/non-covalently bonded to a Heat Shock Protein.

WO2009/150543 describes a conjugated Vi-polysachharide to be used as avaccine composition against Salmonella typhi causing typhoid fever,wherein the Vi-polysaccharide is covalently conjugated to a proteinselected from CRM197 or tetanus toxoid. The method of conjugation asdisclosed in WO2009/150543 includes first simultaneously adding carrierprotein which is preferably CRM197 or tetanus toxoid to a linker such asadipic acid dihydrazide (ADH), and a carbodiimide such as1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDAC), to give aderivatized carrier protein in presence of a 2-(N-morpholino) ethanesulphonic acid (MES buffer). The weight ratio of the carbodiimide EDACto the carrier protein is between 0.1 to 0.15. It also discloses thathigher amounts of carbodiimide/protein ratios can cause aggregateformation. Derivatization of the carrier protein is followed byactivation of the Vi-polysachharide (ViPs) as well. TheVi-polysaccharide is also activated with a carbodiimide wherein variousratios of ViPs and carbodiimide (EDAC) are mixed to activate theVi-polysaccharide. It is mentioned that Vi activation can be performedat room temperature within 2 minutes wherein higher ratios between 1.5:1to 200:1 can be used. The derivatized carrier protein CRM197 or tetanustoxoid and the activated Vi polysaccharide of Salmonella typhi is thenreacted with each other to get the conjugated ViPs-CRM197 or ViPs-TTconjugate, followed by removal of the excess linker.

Safety and immunogenicity of ViPs conjugate vaccines in adults,teenagers and 2- to 4-year old children in Vietnam were evaluated byZuzana Kossaczka et al in1999. In this study the geometric mean level ofanti-Vi-rEPA (conjugate vaccine) in the 2- to 4-year old children washigher than that elicited by Vi capsular polysaccharide vaccine in the 5to 14 years old children. Re-injection of conjugate vaccine induced risein antibody titers in 2- to 4-year old children (T-cell dependent).Konadu et al. (2000) prepared S. paratyphi A O-specific polysaccharide(O-SP) and coupled to tetanus toxoid. These conjugates elicited IgGantibodies in mice and the safety and immunogenicity of the conjugateswas evaluated in Vietnamese adults, teenagers and 2-4 years oldchildren. The study concluded that these experimental conjugates weresafer and proven to elicit IgG antibodies in adults, teenagers and 2-4years old children. The efficacy of Salmonella typhi ViPs conjugatevaccine in two to five-year old children was evaluated by Feng Ying C etal. In this study the conjugate typhoid vaccine was found to be safe andimmunogenic and had more than 90% efficacy in children two to five yearsold. Serum IgG Vi antibodies after six weeks of second dose levelsincreased 10 fold in 36 evaluated children. These cases were followedfor a period of 27 months. No serious adverse reactions were observed inthe study due to the vaccination. Effect of dosage on immunogenicity ofViPs conjugate vaccine injected twice in to 2 to 5 years old Vietnamesechildren was studied by Do Gia Canh et al. In this study dosageimmunogenicity study of 5 μg, 12.5 μg and 25 μg of conjugate vaccineinjected twice, six weeks apart was evaluated. This study also confirmedthe safety and consistent immunogenicity of four lots of conjugatevaccine in this and previous trials. Novartis vaccine institute forglobal health carried-out three different dose-related formulations ofViPs-CRM197. They carried out different doses were 25 μg, 12.5 μg, 5 μgand 1.25 μg/dose. The GMT for these concentration at day 28 was 304U(units), 192U, 111U and 63U respectively. At day 28 GMT with 25 μg/doseelicited the highest antibody level (304U) after single injection.

Although, the present state of the art includes conjugate vaccines withVi-polysaccharide and a carrier protein, however, the existing nativeVi-polysaccharide conjugate vaccines when tested in many human clinicaltrials revealed that these vaccines are safe and immunogenic in adultsbut failed to induce any protective immune response in children below 2years of age. Therefore, this native S. typhi polysaccharide vaccine didnot prove to find any particular solution against deadly S. typhiinfections in children's less than 2 years of age which demands a newvaccine which could immunize children of age below 2 years against S.typhi infections responsible for causing typhoid. The age group of below2 years of age is the most prone to infections by Salmonella typhi butthere seems to be presently not available to the mankind any protectivevaccine against S. typhii for infants below 2 years of age still now. Asdiscussed above, various carrier proteins such as CRM-197, r-EPA, havebeen conjugated to Vi-polysaccharide, wherein the Vi-polysaccharidemight not have been isolated from S. typhi, or being depicted from anyother sources. Producing typhoid conjugate vaccines is therefore,specific to the particular carrier protein involved and the nativepolysaccharide involved in the conjugation process and the resultingconjugate vaccine. Each carrier protein-polysaccharide conjugation makesitself a different identity of conjugate vaccine. The prior artsdisclosed in the area of typhoid conjugate vaccine, methodology and aswell as those currently used, have their own drawbacks, which might be apossible reason behind not having any conjugate vaccine presentlyavailable which can protect children below 2 years of age.

It is also very much evident and well known in the current state of theart that, the present typhoid conjugate vaccines require at least 2 ormore injections with a time interval of 6-8 weeks to comprise a completevaccination schedule A typhoid Vi capsular polysaccharide-tetanus toxoid(ViPs-TT) conjugate vaccine was made available to the public by BioMed,which required 2 injections of 5 μg each with a time interval of 6-8weeks to complete a single vaccination schedule. However, this ViPs-TTvaccine also was not capable to immunize children below 2 years of ageagainst Salmonella Typhii.

Hence, there exists a need of alternating conjugation methodologies,which would reduce costs, and the number of injections to only oneinjection capable of eliciting sufficient immune response and otherassociated technical concerns in the field of conjugation chemistrywhich would be simpler, less time consuming, cost-effective and safe. Anefficient vaccine must be capable of triggering a good immune responseand must be applicable for use in infants especially below 2 years ofage. The disclosure as set forth in this invention attributes to novelalternative methods of conjugating the Vi-polysaccharide along with thespecific carrier protein tetanus toxoid (TT) in an inventive mannerput-forth in this application which potentially overcomes the drawbacksof native polysaccharide vaccines and also current conjugationmethodologies including other ViPs vaccines conjugated to carrierproteins. The Vi-polysaccharide-protein conjugate vaccine produced bythis particular methodology as set forth in this patent applicationmakes it more suitable for immunization in children and infantsincluding less than 2 years of age with secondary memory responsesproducing high affinity antibodies against S. typhi infections,including humans of any age group. It is also another advantage of theinvention put forth in this application that, the number of injectionsof typhoid conjugate vaccine to complete a vaccination schedule has alsobeen reduced to only ONCE, which at the same time elicits a betterimmune response when compared to immune response generated by avaccination schedule of 2 or 3 injections of typhoid conjugate vaccinebeing practiced earlier. Single injection of typhoid conjugate vaccineis always preferable for infants and children since it would reduce,additional visits to the clinic, pain suffered by a child or infant forrepeated injections for vaccination. It is already reported that, 40% ofinjections worldwide are administered with un-sterilized, reusedsyringes and needles, and especially in the targeted developingcountries, this proportion is more than 70%, exposing millions of peopleto infections wherein pathogens enter the tissues of the body during aninjection. Furthermore, poor collection and disposal of dirty injectionequipment, exposes healthcare workers and the community to the risk ofneedle stick injuries. Unfortunately, in some countries, unsafe disposalalso leads to re-sale of used equipment on the black market. Openburning of syringes is unsafe under WHO, yet half of thenon-industrialized countries in the World, follow this practice.(“Injection safety”, Health Topics A to Z. World Health Organization.Retrieved 2011-05-09.). Unsafe injections cause an estimated 1.3 millionearly deaths each year. (M. A. Miller & E. Pisani. “The cost of unsafeinjections”. Bulletin of the World Health Organization 77 (10):1808-811). Although, to improve injection safety, the WHO recommendscertain alternatives to injections subject to availability, or elsecontrolling and regulating the activity of health care workers andpatients, vaccinees, by ensuring the availability of equipment andsupplies aided with managing waste safely and appropriately; thesemeasures are not always possible to be observed absolutely. In suchcircumstances, a combination of Typhoid conjugate and measles vaccine inone SINGLE shot will definitely play a substantial role in decrease ofworries pertaining to injection safety in national immunizationprograms. Many countries do have legislation or policies that mandatethat healthcare professionals use a safety syringe (safety engineeredneedle) or alternative methods of administering medicines wheneverpossible, however reduction in the number of injections for ensuringprotection against Typhoid and Measles in one single injection ininfants surely indicates high compliance from a public healthperspective since where there was at least 3 injections required earlierto inject typhoid (2 injections minimum) and measles (one injection)vaccine, now the same is accomplished by only ONE injection.

OBJECTS OF THE INVENTION

Primary object of the invention is development of a vaccine formulationfor prophylaxis and treatment of Salmonella typhi infections in humansso that the T-independent polysaccharides can be made T-cell dependentthereby facilitating to produce efficient immune responses in childrenof all age groups especially below 2 years of age and also includingadults as well.

Another object of the invention is to provide a vaccine compositionagainst fever caused due to S. typhi with suitable conjugatepolysachharides as the vaccine antigen that would confer protection tochildren below 2 years of age.

One objective of the invention is to provide a fed-batch method ofproduction of Vi capsular polysaccharide.

One more objective of the invention is to provide methods of conjugationof Vi capsular polysaccharide with or without size reduction to acarrier protein.

Yet another objective of the invention is to provide alternative methodseffective conjugation methodology in a reduced time throughsize-reduction of ViPs of Salmonella typhi prior to conjugation withcarrier protein thereby increasing the percentage of conjugation betweenthe Vi polysaccharide and carrier protein.

Yet another object of the invention is to provide a method ofconjugation for Vi capsular polysaccharide of Salmonella typhi and acarrier protein tetanus toxoid as final polysaccharide conjugated bulkand finished vaccine with or without a linker molecule.

A further objective of the invention is to provide immunogenic vaccineformulations comprising coupled polysaccharide-protein conjugates ofVi-polysaccharide-proteins in appropriate single dose and multidosevials in infants and adults to be administered at appropriateconcentrations effective to confer prophylaxis against S. typhi.

Other embodiments and advantages of the invention are set forth in partin the description, which follows, and in part, may be obvious from thisdescription, or may be learned from the practice of the invention.

SUMMARY OF THE INVENTION

According to one embodiment of this invention, cultivation andprocessing of Salmonella typhi Vi-polysaccharide is disclosed. It isfurther purified through several downstream processing steps to obtainpure Vi-polysaccharide.

According to one other embodiment of this invention, method ofconjugation of pure Vi-polysaccharide to conjugate with protein tetanustoxoid is disclosed in the presence of a linker molecule Adipic AcidDihydrazide (ADH). The yield of pure resultant ViPs-TT conjugate is ashigh as 70%-80%.

According to one other alternative embodiment of this invention, methodof conjugation of Vi-polysaccharide to conjugate with protein tetanustoxoid is disclosed without presence of any linker molecule. The yieldof pure resultant ViPs-TT conjugate without linker is as high as70%-80%.

A further embodiment of this invention discloses stable formulations ofViPs-TT conjugate vaccine in appropriate concentrations of ViPs-TT withor without 2-phenoxyehtanol as preservative with ViPs-TT to ensure, acomplete vaccination schedule through one injection only.

One another embodiment of this invention provides, clinicallyestablished experimental data of the stable ViPs-TT conjugate vaccineformulation evidencing strong sero-protection and eliciting the desiredimmunogenecity against Salmonella typhi infections in humans includinginfants below 2 years of age (6 months to 2 years), as well as subjectsin other age groups through only one injection comprising a completevaccination schedule.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1: General flow diagram of ViPs production and conjugation withlinker ADH (left side) and without linker (right side).

FIG. 2: Serological identification test of Vi polysaccharide.

FIG. 3: HPLC (RI Detector) for Typhoid native Vi-polysaccharide, theHP-GPC column profile of the purified Vi-polysaccharide was analyzed byRI detector. The peak at 13.185 minutes represents nativeVi-polysaccharide, which signifies molecular weight of ˜900 kDa.

FIG. 4: Size reduced ViPs using homogenizer for about 45 passes. TheHP-GPC column profile of the size reduced Vi-polysaccharide was analyzedby RI detector. The peak at 16.04 minutes represents size reducedVi-polysaccharide, which signifies molecular weight of ˜200 kDa.

FIG. 5: Size reduced ViPs using microwave oven. The HP-GPC columnprofile of the size reduced Vi-polysaccharide was analyzed by RIdetector. The peak at 15.18 minutes represents size reducedVi-polysaccharide, which signifies molecular weight of ˜250 kDa.

FIG. 6: HPLC (RI) for ViPs-TT conjugate bulk. HPLC Profile of theVi-polysaccharide-Tetanus toxoid conjugate was detected by RI detectorusing HP-GPC column. The peak at 12.83 minutes represents conjugateViPs-TT with linker molecule.

FIG. 7: HPLC (UV) for ViPs-TT conjugate bulk. HPLC Profile of theVi-polysaccharide-Tetanus toxoid conjugate was detected by UV detectorusing HP-GPC column. The peak at 12.66 minutes represents conjugateViPs-TT with linker molecule.

FIG. 8: HPLC (RI) for ViPs-TT conjugate bulk without linker. HPLCprofile of the Vi-polysaccharide-Tetanus toxoid conjugate vaccine wasdetected by RI detector using HP-GPC column. The peak at 12.98 minutesrepresents conjugate ViPs-TT conjugate without linker.

FIG. 9: HPLC (UV) for ViPs-TT conjugate bulk without linker. HPLCprofile of the Vi-polysaccharide-Tetanus toxoid conjugate vaccine wasdetected by UV detector using HP-GPC column. The peak at 12.662 minutesrepresents conjugate ViPs-TT conjugate without linker.

FIG. 10: HPLC (RI) for ViPs-TT conjugate vaccine. HPLC profile of theVi-polysaccharide-Tetanus toxoid conjugate vaccine was detected by RIdetector using HP-GPC column. The peak at 12.82 minutes representsconjugate ViPs-TT conjugate.

FIG. 11: HPLC (UV) for ViPs-TT conjugate vaccine. HPLC profile of theVi-polysaccharide-Tetanus toxoid conjugate vaccine was detected by UVdetector using HP-GPC column The peak at 12.72 minutes representsconjugate ViPs-TT conjugate.

FIG. 12: Comparison of Geometric Mean Titer of different age groupsafter single injection of 25 μg single injection of ViPs-TT conjugatevaccine.

FIG. 13: Comparison of %age seroconversion of different age groups aftersingle injection of 25 μg single injection of ViPs-TT conjugate vaccine.

DETAILED DESCRIPTION OF THE INVENTION

Salmonella typhi are grown in suitable medium and the actively growncells were transferred into the fermenter containing pre-sterilizedmedium. Initially, batch mode fermentation process is carried out andonce the cultures reaches early stationary phases, a feed mediumcontaining high concentration of carbon source was pumped into thefermenter incrementally. Fed-batch mode fermentation process is carriedout till the desired optical density was obtained. The cultures areharvested by inactivating with low concentration of formalin and thencentrifuged to obtain cell supernatant. Hexadecyltrimethylammoniumbromide (Cetavlon) is added to the cell supernatant to precipitate thecrude Vi-polysaccharide from host cell components. Sequentialpurification steps i.e. ethanol precipitations, concentration anddiafiltration using different molecular weight cut-off membranes andsterile filtration techniques were carried out to isolate purifiedVi-polysaccharide from host cell impurities like nucleic acids, proteinsand lipo-polysaccharides.

The factors that influence the coupling of polysaccharides and proteinsdepend upon molecular weight and activation of the functional groups.Low molecular weight of polysaccharides can result in efficientcoupling. Different proteins like tetanus toxoid, diphtheria CRM 197,the B subunit of the heat-labile toxin (LT-B) of Escherichia coli, therecombinant exoprotein A (rEPA) of Pseudomonas aeruginosa, Flagellin FliC, and Horseshoe rab Haemocyanin (HCH) have been mostly used forconjugation. Determining molecular sizes of polysaccharides andpolysaccharide-protein conjugates of bacterial polysaccharides is animportant aspect in designing conjugate vaccines. The assessment ofphysico-chemical characteristics of polysaccharide-protein conjugateplays important role in eliciting specific immune responses.Determination of the molecular size of the polysaccharide before andafter conjugation results in efficient conjugation. The two importantcritical quality control tests employed after conjugation andpurification are the ‘polysaccharide (PS) to protein ratio’ and the‘percent non-conjugated polysaccharide (Free polysaccharide)’.

Podda et al. (2010) reported the epidemiology and significance ofvaccination in the children below two years of age. The currentlyavailable vaccines have some relevant limitations and hence cannot beused in children under two years of age, an age group affected by asignificant burden of typhoid disease. Introduction of a conjugatevaccine is expected to be an effective tool for efficient immunizationof all age groups yet there is no experimental data available at presentwhich would enable vaccination of typhoid conjugate vaccine below 2years of age. This invention, relies on its unique conjugationmethodology of the ViPs-TT conjugate vaccine having an advantage ofmaking it possible to vaccinate children or infants under two years ofage to be prevented from Salmonella typhi infections that causes typhoidfever in this tender age group which is accordingly supported byexperimental clinical trial data, and also reduces the number ofinjections to accomplish a complete vaccination schedule through onlyone dose of the typhoid conjugate vaccine in infants below 2 years ofage.

The following examples illustrate embodiments of the invention, butshould not be viewed as limiting the scope of the invention.

EXAMPLE 1 Cultivation and processing of S. typhi Vi Polysaccharide

The strain Salmonella typhi (Ty2) was obtained from Dr. John Robbins,National institutes of Child Health and Human Development (NICHD), USA.The culture received form NICHD, USA was confirmed and identified asSalmonella serovar typhi by identification of the followingcharacteristics: gram staining, glucose positive without gas formation,H₂S positive on a Xylose Lysine Deoxycholate agar (XLD agar), andpositive serology with Vi-polysaccharide. The purity of the strain wasconfirmed on different selective media such as, Bismuth Sulfite Agar(BSA), Triple Sugar Iron (TSI) agar. The purity of the strain wasconfirmed on different selective media such as Xylose LysineDeoxycholate agar (XLD agar), Bismuth Sulfite Agar (BSA), Triple SugarIron (TSI) agar.

Salmonella typhi Ty2 was grown on Soyabean Casein Digest (SCDM) mediumat 37±1° C., for 12 hours. The bacterial culture was centrifuged and thepellet was re-suspended in sterile glycerol (50%). 0.5 mL aliquots ofthe glycerol suspension in 1 mL cryovials were prepared and stored at-70° C. Viable cell count of the master seed was also carried out. Thecontents of cryovial of the Master seed lot was inoculated into SCDMbroth and incubated at 37±1° C. for 12 hours. The bacterial culture wascentrifuged and the pellet was re-suspended in sterile glycerol (50%).Viable cell count was carried out. Aliquots of the glycerol suspensionin cryovials were prepared and stored at −70° C. The Master and Workingcell banks were characterized by grams staining, utilization of glucose(Durham's method), oxidase test, agglutination test and viable cellcount. This was plated on Tryptone Soya Agar (TSA) and incubated at 37°C. for 48 to 72 hours. Colony count was performed using colony counter.

1.1. Fermentation Process

-   Inoculum development: The contents of one cryovial of the working    seed lot was removed from the freezer and thawed at room temperature    using a water bath. One cryovial from working cell bank of    Salmonella typhi was inoculated into 10 mL Soybean Casein Digest    Medium (SCDM) and cultured at 37±1° C. for 12 hours (Stage-I),    transferred to two flasks each containing 50 mL SCDM at 37±1° C. for    12 hours (Stage-II) and finally transferred to four flasks each    containing 400 mL SCDM and incubated at 37±1° C. for 12 hours    (Stage-III). At every stage of culture transfer, purity and    morphological characteristics was checked by gram staining. The OD    was checked at 600 nm. The OD of the Salmonella typhi culture    recorded at different stages of seed growth varied from 1.2 to 3.8.

Batch Mode Fermentation:

TABLE 1.1 Fermentation parameters and specification limits ParametersRanges pH 6.9 ± 0.2 Dissolved oxygen 70-90% Stirrer speed 250 ± 10 rpmTemperature 37 ± 2° C. Air flow 0.5 ± 0.1 VVM (Volume per volume perminute)

Initially 85 L of SCDM was prepared in 100 L S.S vessel and transferredto the fermentor. This was sterilized in situ at 121° C. for 15 minutes.The medium was cooled to 37° C. At this stage supplement mix was pumpedinto the fermentor through the addition port. To maintain pH, 50%ammonia solution in a bottle was connected to the addition port as anitrogen source. The seed inoculum was transferred into the fermentorand the fermentation process was carried out at a pH of 6.9±0.2,temperature of 37±2° C. and the Dissolved Oxygen is maintained at70-90%. for a period of up to 22 to 24 hours. The growth was checked bytaking the OD values at 600 nm initially at 0 hour and at every 2 hoursup to 24 hours.

-   Fed batch mode fermentation: Fed batch fermentation process for S.    typhi resulted in increased Vi polysaccharide production. Stage III    cultures with an OD₆₀₀ of 3.8 was ideal for fermentation. The pH was    maintained at 6.90 and dissolved oxygen level was between 40% to    60%. To the early stationary phase culture, the feed medium    containing carbon source along with inorganic salts and minerals was    pumped incrementally into the fermenter throughout the fermentation    process. The fed batch fermentation process adopted herein to    increase biomass by feeding with a solution containing glucose at a    range of 1 to 2 mg/mL concentration. The pH was maintained in the    range of 6.90 to 7.20 and dissolved oxygen level was maintained    between 40%-60% Ammonia solution (50%) was supplied as a nitrogen    source along with the feed medium. Foaming was controlled by pumping    antifoam solution through the addition port aseptically. The optimal    pH maintained was 7.2 using 10% NH₄OH and dissolved oxygen    concentration was maintained at 35% air saturation. Glucose level    was monitored every 30 minutes, and through the fed-batch process    the glucose concentration was maintained at about 1 g/L throughout    the process.

Process was continued up to 24 hours and then the bacterial culture wasinactivated with 0.5% formaldehyde and kept under mild stirring inchilled condition (below 15° C.). The growth was checked by taking theOD values at 600 nm for 0 hr and every 2 hours for 24 hours. Thisfeeding strategy resulted in an increase in the biomass in terms ofoptical density to about 120 to 130. The increased biomass translatedinto a greater Vi polysaccharide production which achieved a final yieldof Vi-polysaccharide obtained in the fed batch culture of approx 1000 mgper liter in the present process from which 400 mg of purified ViPS perliter was finally obtained, after completion of downstream processing.Thus, Fed-Batch mode of cultivation resulted in a final yield of atleast 40%.

1.2. Downstream Process of Purified Vi Polysaccharide Bulk (ViPs)

Fermentation cell supernatant is subjected to different steps ofpurification to isolate purified Vi-polysaccharide. Vi-polysaccharideconsists of partly 3-O-acetylated repeated units of2-acetylamino-2-deoxy-d-galactopyranuronic acid with α-(1→4) linkages.Hence the determination of O-acetyl content could be correlated to theamount of Vi-polysaccharide. The final pure Vi polysaccharide fractionshould contain 2 mM of O acetyl per gram of Vi-polysaccharide (WHO TRS840). The supernatant normally contains large amount of proteins,nucleic acid and lipopolysaccharides. Filtration techniques play animportant role in downstream processing in purification of bacterialpolysaccharides from host cell impurities. Retention of the desiredmolecule from the dissolved substances is done on the basis of size;higher sized particles will be retained at the surface and those lowerthan the nominal weight limit (NMWL) of the membrane flow out in thepermeate (Jagannathan et al., 2008). 100 kDa cut-off membrane cassetteswere used at initial step of cell supernatant concentration and 300 kDacut-off membrane cassettes at final concentration step and diafilteredusing sterile water for injection (WFI).

-   Cell separation: The harvested culture was centrifuged in a bowl    centrifuge at 9000 rpm (8000 g) for 30 minutes at 4° C. The    supernatant was collected in sterile vessels. A sample was taken    from the supernatant and assayed for O-acetyl content.-   Concentration and diafiltration: The supernatant was diafiltered by    using tangential flow filtration (TFF) system using 100 kDa    membrane. The supernatant was concentrated to 1/10^(th) of the    original volume and further diafiltered with water for injection    (WFI) till the required concentrate was obtained. O-acetyl content    of the concentrate was assayed.-   Cetrimide precipitation: To the concentrate 0.4 M cetrimide was    added and incubated at (5°±1° C.) for 3±1 hours. The contents were    centrifuged at 9000 rpm for 30 minutes at 4° C. The pellet collected    was suspended in the required volume of 1 M NaCl. The O-acetyl    content of the pellet suspension was determined.-   Ethanol precipitation: One volume of ethanol and 2% of sodium    acetate were added to the resuspended cetrimide precipitate; the    contents were stirred for 20±5 minutes using a magnetic stirrer.    Contents were centrifuged at 4200 rpm (8000 g) for 30 minutes at    4° C. The supernatant was collected into a sterile bottle and the    pellet was discarded. To the supernatant, two volumes of ethanol    were added (100%) under continuous stirring for a period of 60±10    minutes. 2% of sodium acetate was added to the above content under    continuous stirring. After 1 hour of incubation, the contents were    centrifuged at 4200 rpm (8000 g) for 30 minutes at 4° C. The    supernatant was discarded; pellet was suspended in sterile cool WFI    and transferred to sterile bottle. Sample was checked for O acetyl    content.-   Filtration: The concentrated ViPs bulk was passed through 0.22 μ    capsule filter (Sartopore, Sartorius). This sterile filtered    purified bulk of ViPs was assayed for O-acetyl content. The ViPs    bulk thus obtained was re-extracted with cetrimide and precipitated    with ethanol. Finally, the bulk was concentrated and diafiltered    using a 300 kDa cassette (known as concentrated bulk) as mentioned    above. The O-acetyl content was assayed after each process. The    following O-acetyl contents at different steps of downstream    processing, as given in the table 1.2 below was obtained. The    O-acetyl content was analyzed by Hestrin method as described below.-   Assay for O-acetyl content: Determination of O-acetyl content was    performed by the method of Hestrin. (Hestrin,1949). The amount    O-acetyl in the sample was proportional to the amount of Vi-content    expressed in mg/mL. 0.5 mL of 3.6 N HCl and 1 mL of alkaline    hydroxylamine solution were added to the test samples and mixed    thoroughly. The mixture was kept at room temperature for 2 minutes    and 0.5 mL of ferric chloride solution added and mixed well. The    absorbance was measured at 540 nm. The O-acetyl content was    calculated as follows:

${O\text{-}{{acetyl}\left( {{\mu moles}/{mL}} \right)}} = \frac{{Test}\mspace{14mu} {OD} \times {Standard}\mspace{14mu} {concentration} \times {dilution}\mspace{14mu} {factor}}{{Standard}\mspace{14mu} {OD}}$Factor  for  O-acetyl  to  Vi  content  conversion = O-acetyl  (μmoles/mL) × 0.294(25/0.085/1000) = Vi  content  (mg/mL)

The final sterile filtered (0.22 μ) Vi-polysaccharide bulk islyophilized in a low temperature vacuum dryer (Lyophilizer—FTS system).The lyophilized powder was tested for serological identification byOuchterlony method, moisture content, protein content, nucleic acids,molecular size distribution and bacterial endotoxin content. In thepresent study purified Vi-polysaccharide and corresponding homologousantisera were filled in the wells until the meniscus just disappears.The gel plate was incubated in a humidity chamber. The precipitin lineswere observed by naked eye when the plate was seen against a brightlight back ground. A photograph of the same is shown in FIG. 2, showinga clear precipitin arc observed.

The molecular size distribution of Vi-polysaccharide was determined byusing gel permeation column with Sepharose CL-4B as stationary phase.Fractions were collected after void volume (Vo) corresponding to kDa0.25 and pooled together. 75% of poly-saccharide eluted at kDa of 0.25.The molecular size distribution of S. typhi Vi-polysaccharide bulk isgiven in the Table 4.5 below. Characterization of Vi revealed it to have1% nucleic acids, 0.3% of proteins and an O-acetylation of level of 86%by H-NMR.

Results of dried ViPs bulk obtained for a single batch are tabulated inthe Table 1.2 below:

TABLE 1.2 Results of dried Vi-polysaccharide bulk Tests ResultsSerological identification Clear precipitin arc was observed(Ouchterlony) Moisture content 1.80% Protein 2.5 mg/g of Vipolysaccharide powder Nucleic acids 5 mg/g of Vi polysaccharide powderO-acetyl content (Hestrin) 2.1 mmoles/g of Vi polysaccharide powderMolecular size distribution 75% of polysaccharide eluted at 0.25 kDaEndotoxins Less than 150 EU/μg of Vi-polysaccharide powder

The above results met all the requirements of WHO TRS 840, Britishpharmacopeia (2010) and Indian pharmacopeia (2010) standards. Therequirements of WHO TRS 840 (1994) were considered as standardspecifications in present study. The standard requirements of WHO areproteins 10 mg/g, nucleic acids 20 mg/g, O-acetyl content not less than2 mmol/g of Vi-polysaccharide, molecular size of 50% polysaccharideshould elute before 0.25 kDa, Identity by immune precipitation methodand sterility test passing. Accordingly, to the British and Europeanpharmacopia (2007), the dried Vi-polysaccharide specifications are:protein 10 mg/g, nucleic acids 10 mg/g, O-acetyl groups 2 mmol/g, notless than 50 per cent of the polysaccharide to be found in the poolcontaining fractions eluted before kDa 0.25, identification using aimmunoprecipitation method, and bacterial endotoxin test. Thesespecifications are similar to the WHO TRS 840, British pharmacopoeia(2010) and Indian pharmacopoeia (2010).

EXAMPLE 2 Conjugation Methodology

Efficient methods of conjugation of the purified Vi polysaccharide(ViPs) to a carrier protein selected from any bacterial protein or aviral protein, such as diphtheria toxoid, tetanus toxoid, Pseudomonasaeruginosa toxoid, pertusis toxoid, Clostridium perfringens toxoid,Pseudomonas exoprotein A, Flagellin Fli C, and CRM197 are disclosed inthis present invention. Preferably the purified ViPs is conjugated totetanus toxoid in this present invention. High yield of conjugation isachieved employing various alternative conjugation methodologies. Thepurified ViPs may be subjected for size reduction prior to conjugation.In the present invention, efficiency of conjugation using either highmolecular size (non size reduced) or low molecular size (size-reduced)ViPs was conducted in both the methodologies to achieve high yields ofpurified ViPs-TT conjugate. For conjugation with high molecular sizeViPs and tetanus toxoid, the concentration of ViPs (non-size reduced) inthe final reaction mixture shall lie in the range of 1 mg/ml to 10 mg/mlto obtain the desired yields of ViPs-TT conjugate upto 70%-80%, whereasfor conjugation of low molecular size ViPs and tetanus toxoid, theconcentration of ViPs (size reduced) in the final reaction mixture shalllie in the range of 5 mg/ml to 10 mg/ml to obtain the desired yields ofViPs-TT upto 70%-80%. Alternative methods of size reduction of thepurified ViPs is disclosed in the following sections.

The novelty of the present invention is modification of the Vipolysaccharide and activating them with a linker or without a linkermolecule in presence of cross linking agents. According to the presentinvention, there lies no requirement to activate conjugate proteins.Conjugation between activated polysachharides and carrier proteins takesplace in presence of cross linking agents such as EDAC. WO2009/150543teaches derivatizing the proteins for conjugation, in which the Vipolysaccharide was isolated from C. freundi and further conjugated withCRM197 and/or tetanus toxoid as carrier proteins. In their study Vi andEDAC were mixed at appropriate molar ratio (EDAC/Vi) of 0.9-1.4,alternatively CRM197 and/or TT were derivatized with treatment with ADHand EDAC. Vi was conjugated to CRM197 and TT separately and theconjugation mixture was purified using Sephacryl S-1000; fractions wereanalysed by SDS-PAGE and those which did not contain free protein werecollected (Micoli et al., 2011). However, according to WO2009/150543,the excess linker has been removed by dialysis, whereas in the presentinvention, Vi-polysaccharide was optionally subjected for size reduction(homogenization or by microwave method) and then conjugation has beenachieved optionally coupled to the linker molecule or without any linkermolecule at all. Hence, wherein linker molecule has not been used, thereis no requirement of additional step of removing excess linker molecule.Additionally, in the process involving conjugation with the linkermolecule, excess linker was removed by desalting and diafiltrationunlike dialysis as mentioned in WO2009/150543.

2.1. Size Reduction of ViPs using High Pressure Homogenization

ViPs is a very large molecule of nearly 1000 kDa. Therefore, the size ofthe molecule is preferably reduced to approximately one fourth of thelarge molecule for enabling conjugation with carrier proteins includingTetanus Toxoid at low concentrations. Therefore, the ViPs at aconcentration of 5-7.5 mg/ml was subjected to high pressurehomogenization at 1500 bar at 2-8° C. and the same activity was repeatedfor at least 45 passes. The molecular size of the reduced ViPs wasthereafter verified through Size Exclusion-Gel Permeation Chromatographyas shown in corresponding figures. The retention time of ViPs beforesize exclusion was 13.185 minutes (FIG. 3), whereas after size exclusionchromatography the retention time of ViPs was eluted at 16.04 minute(FIG. 4), which signifies that the ViPs has been reduced to acorresponding molecular size of approximately 200 kDa. The O-acetylcontent of the size reduced ViPs remains the same after homogenizationtreatment verified by hestrin method. Thereafter, the size reduced ViPswas subjected further to subsequent conjugation steps as discussed inthe following sections.

2.2. Size Reduction of ViPs using Microwave Oven

Another method of size reduction of ViPs prior to conjugation was doneusing micro-wave oven. The ViPs at a concentration of 5-7.5 mg/ml in aglass bottle was put inside a micro-wave oven at 50%-100% power for 5-10minutes. The micro waves generated inside the oven is responsible forcleaving the glycosyl bonds of long chains of the Vi polysachharide toreduce it to shorter molecules required to conjugate them to carrierprotein. The molecular size of the reduced ViPs was thereafter verifiedthrough Size Exclusion-Gel Permeation Chromatography as shown in thecorresponding figures. The retention time of ViPs before size exclusionwas 13.185 minutes (FIG. 3) whereas after size exclusion chromatographythe retention time of ViPs was eluted at 15.18 minute (FIG. 5), whichsignifies that the ViPs has been reduced to a corresponding molecularsize of approximately 250 kDa. The O-acetyl content of the size reducedViPs remains the same after microwave treatment verified by hestrinmethod. Thereafter, the size reduced ViPs was subjected furtherconjugation techniques as discussed in the following sections.

2.3. Conjugation of Vi Polysaccharide and Tetanus Toxoid with a Linker

The purified Vi polysaccharide (either size reduced or non-size reduced)were partially de-O-acetylated in presence of sodium bicarbonate, andcoupled with ADH using EDAC mediated reaction at a range of pH 6.0-7.5.The reaction was maintained at 2-8° C. with mild stirring. Afterincubation, the reaction mixture was quenched by bringing the pH to 8.0using phosphate buffer-EDTA buffer and further dialyzed using lowmolecular cut-off membranes with initially phosphate and then followedby MES buffer. The final mixture is concentrated and tested for O-acetylcontent, ViPs-ADH ratio, free ADH.

The tetanus toxoid was concentrated and diafiltered with MES bufferusing low molecular weight cut off membrane. The final concentratedTetanus toxoid is tested for protein content. For conjugation themodified Vi-polysaccharides and proteins are coupled in the presence ofcarbodiimide condensation using EDAC. The final coupled molecules areconcentrated and diafiltered using a 1000 kDa cut-off membranepreferably PES (polyether sulphone) membrane, followed by continuousbuffer exchange using 20 diavolumes of phosphate buffered saline. Theretentate which contained purified ViPs-TT is checked forpolysaccharide-protein ratio which shall be within the ratio of 0.5% to1.5%, Vi-content, protein content and molecular size distribution. Finalconjugate bulk was sterile filtered using 0.22 μ membrane and stored at2-8° C.

Optionally, the final coupled molecules are concentrated and diafilteredusing a 1000 kDa cut-off membrane preferably PES (polyether sulphone)membrane, using phosphate buffered saline and then loaded into a gelpermeation column (Sepharose cross linked beads). Fractions collectedwhich are within the ratio of 0.5% to 1.5% were pooled together,concentrated and checked for polysaccharide-protein ratio, Vi-content,protein content and molecular size distribution. Final conjugate bulkwas sterile filtered using 0.22 μ membrane and stored at 2-8° C.

The molecular size distribution of the present invention, Vipolysaccharide conjugate bulk is given in the Table 2.1. The molecularsize of the ViPs-TT conjugate obtained in the present invention is 0.3kDa; when compared with the results obtained in earlier studies of theconjugate ViPs-TT the molecular size distribution of the given conjugatewas less than 0.1 kDa. This means, molecular size distribution of 0.3kDa indicates optimal filterable size which allows proper filtration ofthe ViPs-TT, at the same time providing better immunogenecity to theconjugate vaccine as compared to other lower molecular sizedistribution(s) provided in the prior arts. Bigger molecular sizesignifies better immunogenecity, whereas it is also essential to limitthe molecular size, at appropriate size which would allow filtration ofthe ViPs-TT. Therefore, due to this molecular size distribution of 0.3kDa only ONE single injection of the typhoid conjugate vaccine as laiddown in this present invention, is sufficient to comprise a completevaccination schedule against typhoid fever caused by Salmonella typhi.Prior art prescribes more than one injection, preferably three doses incase of lower molecular size distribution conjugate vaccines againsttyphoid fever.

Determination of total and free (unbound) Vi polysaccharide was measuredby HPAEC-PAD analysis. In the present methodology the Vi conjugate hasyielded 75% of Vi polysaccharide conjugate as eluted at kDa 0.30 therebygiving better polydispersity, and yielded Vi content 0.56 mg/ml, freeViPs 5%, protein content 0.25 mg/mL, Vi Ps-Protein ratio-1.05, freeprotein peak not detectable and sterility was found be acceptable (ReferTable 2.1). The present methodology was performed with an initial batchsize of 10 gms of ViPs, which yielded 8 liters of ViPs conjugate bulk ata Vi conjugate concentration of 0.9 mg/ml-1.0 mg/ml which yielded 7-8gms of ViPs-TT conjugate, thereby giving a yield of 70%-80%.

TABLE 2.1 Results of the ViPs-TT conjugate bulk with linker TestsResults (in ranges) Molecular size 75.7% of polysaccharide eluted at kDa0.3 distribution Conjugate Vi content 0.9 mg/ml-1.0 mg/ml Free Vi Ps 3%-6%. Protein content 0.78 mg/ml-0.9 mg/ml  Vi Ps/protein ratio 1.1Free protein Peak was not detectable at 17^(th)-18^(th) minute in HPLCUV (280 nm) chromatogram. Free protein is absent Sterility No growth wasobserved

FIGS. 6 to 7 represents HPLC Chromatograms of Vi-polysaccharide, andViPs-TT Conjugate bulk at different stages with linker. All the givenHPLC profiles clearly demonstrate the conjugation efficiency of thepresent methodologies.

The conjugation methodology with linker molecule ADH to obtain apurified ViPs-TT conjugate vaccine antigen for preparation of aconjugate vaccine formulation against typhoid fever caused by Salmonellatyphi as described above can be summarized with the following steps:

-   -   a. Fed-batch mode of cultivation to obtain purified ViPs with a        feed medium, the said feed medium comprising feeding with a        solution containing glucose at a range of 1 to 2 mg/mL        concentration at a pH maintained in the range of 6.90 to 7.20        and dissolved oxygen level maintained between 40%-60%, wherein        ammonia solution (50%) was supplied as a nitrogen source along        with the feed medium;    -   b. optionally size reduction of ViPs, wherein the ViPs at a        concentration of 5-7.5 mg/ml is subjected to high pressure        homogenization at 1500 bar at 2-8° C. and the same activity        repeated for at least 45 passes or by a microwave oven so that        to a corresponding molecular size of purified ViPs of        approximately 250 kDa is obtained;    -   c. treating the purified ViPs of step (a) or step (b) with a        cross linking agent EDAC;    -   d. activating the ViPs of step (c) with a linker molecule ADH in        presence of EDAC;    -   e. treating the activated ViPs linked to a linker molecule ADH        of step (d) at a concentration of 1 mg/ml to 5 mg/ml of purified        ViPs of ˜900 kDa or at a concentration of 5 mg/ml to 7.5 mg/ml        of purified ViPs of ˜250 kDa with a carrier protein in presence        of EDAC to form the Vi-polysaccharide-carrier protein conjugate;    -   f. diafiltering through continuous buffer exchange with        phosphate buffered saline of the Vi-polysaccharide-carrier        protein conjugate of step (e) with a 1000 kDa membrane to obtain        the purified ViPs-carrier protein vaccine antigen.        2.4. Conjugation of Vi-Polysaccharide and Tetanus Toxoid without        a Linker

The purified Vi polysaccharide (either size reduced or non-size reduced)were taken in the buffer of MES (2-morpholino ethane sulphonic acid), orPBS, or in physiological saline, at a pH varying from 5.0 to 9.0 (exactpH 6-7.5), the concentration of polysaccharide varies from 1.0 mg to 20mg/ml (5 mg/ml). The protein was taken in the buffer like, MES, or PBS,or in physiological saline at a pH varying from 6.0 to 9.0 (exact pH6-7.5), at a different concentration of 2.0 mg/ml to 20.mg/ml (10mg/ml). Ratio of ViPs to protein should be between 1:1 to 1:3 meaningthereby if a total of 1 gm of ViPs is taken, then equivalent of 1 gm to3 gm protein shall be subjected for conjugation. Conjugation wasperformed at 2° C.-8° C., to control the reaction rate effectively ascompared to room temperature. At higher temperatures, the rate ofconjugation is very fast. It is not preferable to expose polysachharidesto higher temperatures, since, after forming conjugates at highertemperatures, there lies possibilities of aggregation of the conjugatedpolysaccharides-protein molecules. This will increase the size of themolecules, which will become a difficulty to further purify theconjugate proteins in the subsequent steps. Hence, the conjugation ispreferred at 2-8° C. The ViPs and TT were added together at a differentconcentration in any of the buffers described above at different pHconditions and incubated for conjugation. The time of incubation variesbetween 15-45 minutes at room temperature (25° C.), and within 1 hour to2 hours at 2-8° C., whereas while following conjugation methodologyusing ADH (with linker), the incubation time required for conjugation isminimum 2-4 hrs at 2° C. to 8° C. Therefore, the total reaction time isalso reduced following this method of conjugation without linkercompared to conjugation with linker (FIG. 1).

The final coupled molecules are concentrated and diafiltered using a1000 kDa cut-off membrane preferably PES (polyether sulphone) membrane,followed by continuous buffer exchange using 20 diavolumes of phosphatebuffered saline. The retentate which contained purified ViPs-TT ischecked for polysaccharide-protein ratio which shall be within the ratioof 0.5% to 1.5%, Vi-content, protein content and molecular sizedistribution. Final conjugate bulk was sterile filtered using 0.22umembrane and stored at 2-8° C.

Optionally, the final coupled molecules are concentrated and diafilteredusing a 1000 kDa cut-off membrane preferably PES (polyether sulphone)membrane, using phosphate buffered saline and then loaded into a gelpermeation column (Sepharose cross linked beads). Fractions collectedwhich are within the ratio of 0.5% to 1.5% were pooled together,concentrated and checked for polysaccharide-protein ratio, Vi-content,protein content and molecular size distribution. Final conjugate bulkwas sterile filtered using 0.22 μ membrane and stored at 2-8° C.

The present conjugation methodology without any linker molecule wasperformed with an initial batch size of 10 gms of ViPs, which yielded 8liters of ViPs conjugate bulk at a Vi conjugate concentration of 0.9mg/ml-1.0 mg/ml which yielded 7-8 gms of ViPs-TT conjugate, therebygiving a yield of 70%-80%.

TABLE 2.2 Results of the ViPs-TT conjugate bulk without linker TestsResults (in ranges) Molecular size 74.3% of polysaccharide eluted at kDa0.3 distribution Conjugate Vi content 0.9 mg/ml-1.0 mg/ml Free Vi Ps 3%-6%. Protein content 0.75 mg/ml-0.8 mg/ml  Vi Ps/protein ratio 1.2Free protein Peak was not detectable at 17^(th)-18^(th) minute in HPLCUV (280 nm) chromatogram. Free protein is absent Sterility No growth wasobserved

The crude conjugate then obtained is purified by GPC, TFF, Ion Exchangeor HIC. The conjugate matches all required specifications ofpharmacopeia and further sterile filtered. FIGS. 8 to 9 represents HPLCChromatograms of Vi-polysaccharide, and ViPs-TT Conjugate bulk atdifferent stages without linker. All the given HPLC profiles clearlydemonstrate the conjugation efficiency of the present methodologies(FIGS. 10 and 11).

The conjugation methodology without any linker molecule ADH to obtain apurified ViPs-TT conjugate vaccine antigen for preparation of aconjugate vaccine formulation against typhoid fever caused by Salmonellatyphi as described above can be summarized with the following steps:

-   -   a. Fed-batch mode of cultivation to obtain purified ViPs with a        feed medium, the feed medium comprising feeding a solution        containing glucose at a range of 1 to 2 mg/mL concentration at a        pH maintained in the range of 6.90 to 7.20 and dissolved oxygen        level maintained between 40%-60%, wherein ammonia solution (50%)        was supplied as a nitrogen source along with the feed medium;    -   b. optionally size reduction of ViPs, wherein the ViPs at a        concentration of 5-7.5 mg/ml is subjected to high pressure        homogenization at 1500 bar at 2-8° C. and the same activity        repeated for at least 45 passes or by a microwave oven so that        to a corresponding molecular size of purified ViPs of        approximately 250 kDa is obtained;    -   c. treating the purified ViPs of step (a) or step (b) with a        cross linking agent EDAC;    -   d. treating the carrier protein with the ViPs of step (c) at a        concentration of 1 mg/ml to 5 mg/ml of purified ViPs of ˜900 kDa        or at a concentration of 5mg/ml to 7.5 mg/ml of purified ViPs of        ˜250 kDa in presence of a cross linking agent EDAC to form the        Vi-polysaccharide-carrier protein conjugate;    -   e. diafiltering through continuous buffer exchange with        phosphate buffered saline of the ViPs-carrier protein conjugate        of step (d) with a 1000 kDa membrane to obtain the purified        ViPs-carrier protein vaccine antigen.

A linker molecule for example ADH, contains terminal amine groups atboth the ends. The Vi native polysaccharide which is further reduced inits size prior to conjugation, contains abundant functional carboxylgroups (—COOH) naturally. Carrier proteins for example, tetanus toxoidcontain both the amine (—NH₂) and the carboxyl groups (—COOH). In caseof conjugation of the ViPs to the carrier protein with the help of alinker molecule ADH, is effected in presence of cross linking agentssuch as EDAC, wherein the —COOH group of the ViPs should bind with theone —NH₂ group of the ADH linker through one of its ends. The activatedViPs is coupled with the linker ADH, connected through a —CONH bond atone end of the ADH molecule. The other end of the ADH molecule remainsfree to be further bond with the —COOH group present in the carrierproteins at appropriate concentrations and temperature ranges. Theactivated ViPs-ADH is therefore again reacted with the carrier proteinin presence of cross linking agent EDAC, which enables the —NH₂ presentat the other end of the ADH molecule to bind with the —COOH group of thecarrier protein molecule, thereby forming an effective bridge betweenthe Vi-polysaccharide and the carrier protein. Thus in this method,there is a necessity remove excess linkers, after treating ViPs withADH, and again after treating ViPs-ADH to carrier protein. Further EDACis required to use twice in this method.

On the other hand, while following the methodology of conjugating ViPswith the carrier proteins without any linker molecule, since ViPs hasfree —COOH groups and the carrier proteins have free —NH₂ groups, it ispossible to directly bond the —COOH of ViPs to the —NH₂ of the carrierproteins through treatment in presence of cross linking agents such asEDAC. The whole reaction is carried out within one step, which minimizesexcessive use of EDAC as well as reduces the time to accomplishconjugation of ViPs to the carrier protein. Since all carrier proteinscontain free —NH₂ groups, and ViPs also possesses free —COOH, it ispossible to conjugate any carrier protein for example diphtheria toxoid,tetanus toxoid, CRM197 etc with Vi-polysaccharide through this method.Thus, there lies no requirement of using any linker molecule (ADH) forconjugating the ViPs to the carrier protein. The advantage ofconjugation without linker reflects in the stability of the conjugates,because of absence of any connecting molecular bridges between the ViPsand the carrier protein through ADH. This ensures better stability dueto the improved strength of the ViPs-carrier protein conjugate (ViPs-TTin this case) molecule in absence of any connecting bridges. Furtherdegradation of the ViPs-TT is also reduced to very high extent. Also inthis method, it is fairly easy to handle and carry out theexperimentation. The total amount of EDAC required is lesser to about50%, and handled only once instead of using twice in case of ADH linkermethod. (EDAC is an irritant potential of causing protein coagulation onprolonged exposure). Additionally, there is no requirement of GPC columnor TFF system to remove excessive linkers. As the number of steps arereduced, we can minimize the loss of ViPs meant for conjugation to anycarrier protein (for example TT), since the purification stepspertaining to ViPs-ADH linking are omitted. The following tableexemplifies the completion of the entire conjugation experiment withreduced steps and the total time taken in comparison with and withoutlinker molecule.

Total Time Taken in the Whole Conjugation Experiment

TABLE 2.3 Comparison of time taken to complete the conjugation processExperiment with ADH linker Experiment without ADH linker Activity Timetaken Activity Time Taken Reaction of ViPs with  4 hrs Not required Notapplicable ADH Removal of free ADH 12-15 hrs   Not required Notapplicable Analysis of % age ADH  2 hrs Not required Not applicablelinked to ViPs Reaction of ViPs with TT 2-4 hrs at 2° C. to 8° C.Reaction of ViPs with TT 1-2 hrs at 2° C. to 8° C. Purification ofViPs-TT 10 hrs Purification of ViPs-TT 10 hrs conjugate conjugateViPs-TT Fraction 10 hrs ViPs-TT Fraction 10 hrs Analysis AnalysisPooling and Sterile  3 hrs Pooling and Sterile  3 hrs filtrationfiltration Final conjugate analysis  2 hrs Final conjugate analysis  2hrs (HPLC, Vi-content, (HPLC, Vi-content, protein content, ratio)protein content, ratio) Total time taken 45-50 hrs   Total time taken25-27 hrs  

EXAMPLE 3 Vaccine Formulation and Stability

A typical single dose of the typhoid conjugate vaccine formulationclaimed under this invention comprises of Vi-TT conjugate as antigenfrom 15 microgram (μg) to 25 μg dissolved in normal saline made up to atotal volume of 0.5 ml for one injection for a complete vaccinationschedule.

The vaccine formulation as claimed under this invention is also madeavailable in the form of multi-dose vials. Multi-dose vials may beeither of 5 doses (for 5 different vaccinees/subjects/intended vaccinerecipients), or 10 doses (for 10 different vaccinees/subjects/intendedvaccine recipients). In case of multi-dose vials, preservatives areadded to the vaccine formulation to avoid contamination of the vaccineformulation for multiple pricking of the vial in order to vaccinate 5-10different children from the same vaccine multi-dose vial. The multi-dosevials of ViPs-TT typhoid conjugate vaccine formulation of the presentinvention uses a unique preservative 2-phenoxy ethanol, which is freefrom mercury chloride and thiomersal. Disadvantages of usingconventional preservatives such as mercuric chloride and thiomersalcontributing to carcinogenicity has been reported in the current stateof the art. Therefore, use of this unique preservative 2-phenoxy ethanolovercomes the disadvantages of the conventional preservatives mercuricchloride and thiomersal. The details of the multidose vials and theirformulation is tabulated below:

TABLE 3.1 Vaccine formulation of single dose and multidose vials Vaccinecomponent Single dose 5 dose multi vial 10 dose multi vial Vi-TTconjugate 15 μg to 25 μg 75 μg to 125 μg 150 μg to 250 μg PreservativeNot required 25 mg (10% v/v) 50 mg (10% v/v) 2-phenoxy ethanol Normalsaline Quantity Quantity Quantity sufficient sufficient sufficient DoseVolume 0.5 ml 2.5 ml 5.0 ml

The stability of the ViPs-TT conjugate vaccine of BBIL has been studiedand confirmed in detail for 3 years. The ViPs typhoid conjugate ViPs-TTvaccine was subjected for stability study of both accelerated storageconditions (25° C.±2° C.) for 6 months and real time storage conditions(2° C. to 8° C.) for 36 months and found that the test results obtainedare within the limits and complies for the required specification (Table3.2 to 3.5).

TABLE 3.2 Stability study of Typbar-TCV ™ (conjugated with linkermolecule) at 2° C. to 8° C. (25 μg per dose Vi-TT of 0.5 ml) AbnormalTest for Toxicity Description Pyrogens All A Clear, Summed AnimalsColourless responses must liquid, free Identification O-acetyl of 3Survive Sterility from (Ouchterlony) content Vi rabbits for seven Shouldvisible Clear pH (Hestrin) content Free should days and complyparticles. precipitation 6.50 0.064 to (Assay) ViPs not show no with theby visual arc should be to 0.106 μmoles/ 20-30 μg/ NMT exceed weightTest for Time observation observed 7.50 dose dose 20% 1.15° C. LossSterility Zero Complies Complies 7.08 0.099 29.30 6.3 0.6 CompliesComplies day 3^(rd) Complies Complies 7.09 0.098 28.91 6.2 0.5 CompliesComplies month 6^(th) Complies Complies 7.15 0.093 28.45 5.9 0.6Complies Complies month 9^(th) Complies Complies 7.13 0.094 28.31 6.30.6 Complies Complies month 12^(th) Complies Complies 7.03 0.091 27.896.3 0.6 Complies Complies month 18^(th) Complies Complies 7.06 0.08727.45 6.1 0.5 Complies Complies month 24^(th) Complies Complies 7.150.089 27.16 5.7 0.6 Complies Complies month 36^(th) Complies Complies7.02 0.080 26.56 6.0 0.5 Complies Complies month

TABLE 3.3 Stability study of Typbar-TCV ™ (conjugated with linkermolecule) at 25° C. ± 2° C. (25 μg per dose Vi-TT of 0.5 ml) AbnormalTest for Toxicity Description Pyrogens All A Clear, Summed AnimalsColourless responses must liquid, free Identification O-acetyl of 3Survive Sterility from (Ouchterlony) content Vi rabbits for seven Shouldvisible Clear pH (Hestrin) content Free should days and complyparticles. precipitation 6.50 0.064 to (Assay) ViPs not show no with theby visual arc should be to 0.106 μmoles/ 20-30 μg/ NMT exceed weightTest for Time observation observed 7.50 dose dose 20% 1.15° C. LossSterility Zero Complies Complies 7.15 0.098 28.82 4.5 0.3 CompliesComplies day 1^(st) Complies Complies 7.13 0.097 28.52 4.1 0.4 CompliesComplies month 2^(nd) Complies Complies 7.16 0.095 27.94 4.4 0.5Complies Complies month 3^(rd) Complies Complies 7.12 0.093 27.35 4.90.5 Complies Complies month 6^(th) Complies Complies 7.10 0.092 26.8 5.30.4 Complies Complies month

TABLE 3.4 Stability study of Typbar-TCV ™ (conjugated with linkermolecule) at 2° C. to 8° C. (25 μg per dose Vi-TT of 0.5 ml) AbnormalTest for Toxicity Description Pyrogens All A Clear, Summed AnimalsColourless responses must liquid, free Identification O-acetyl of 3Survive Sterility from (Ouchterlony) content Vi rabbits for seven Shouldvisible Clear pH (Hestrin) content Free should days and complyparticles. precipitation 6.50 0.064 to (Assay) ViPs not show no with theby visual arc should be to 0.106 μmoles/ 20-30 μg/ NMT exceed weightTest for Time observation observed 7.50 dose dose 20% 1.15° C. LossSterility Zero Complies Complies 7.03 0.101 29.60 6.0 0.5 CompliesComplies day 3^(rd) Complies Complies 7.05 0.095 27.93 6.3 0.7 CompliesComplies month 6^(th) Complies Complies 7.15 0.093 27.34 5.8 0.5Complies Complies month 9^(th) Complies Complies 7.10 0.094 27.63 6.00.5 Complies Complies month 12^(th) Complies Complies 7.00 0.092 27.046.3 0.6 Complies Complies month 18^(th) Complies Complies 7.02 0.08625.28 6.2 0.6 Complies Complies month 24^(th) Complies Complies 7.110.087 25.57 6.5 0.7 Complies Complies month 36^(th) Complies Complies7.04 0.086 25.28 6.7 0.5 Complies Complies month

TABLE 3.5 Stability study of Typbar-TCV ™ (conjugated with linkermolecule) at 25° C. ± 2° C. (25 μg per dose Vi-TT of 0.5 ml) AbnormalTest for Toxicity Description Pyrogens All A Clear, Summed AnimalsColourless responses must liquid, free Identification O-acetyl of 3Survive Sterility from (Ouchterlony) content Vi rabbits for seven Shouldvisible Clear pH (Hestrin) content Free should days and complyparticles. precipitation 6.50 0.064 to (Assay) ViPs not show no with theby visual arc should be to 0.106 μmoles/ 20-30 μg/ NMT exceed weightTest for Time observation observed 7.50 dose dose 20% 1.15° C. LossSterility Zero Complies Complies 7.10 0.093 27.30 5.0 0.3 CompliesComplies day 1^(st) Complies Complies 7.12 0.095 27.93 4.9 0.6 CompliesComplies month 2^(nd) Complies Complies 7.15 0.098 28.80 5.1 0.4Complies Complies month 3^(rd) Complies Complies 7.13 0.094 27.63 5.30.5 Complies Complies month 6^(th) Complies Complies 7.11 0.095 27.935.7 0.6 Complies Complies month

EXAMPLE 4 Clinical Trials

The final Vi-polysaccharide-tetanus toxoid conjugate bulks wereformulated and tested for immunogenicity in Balb/c mice in comparisonwith native polysaccharide vaccine. Challenge study was carried toassess protective efficacy of the vaccine and preclinical trial wascarried to ensure abnormal, acute and systemic toxicity in laboratoryanimals. Further, the effectiveness of the test vaccine Vi capsularpolysaccharide-tetanus toxoid conjugate (Vi-TT) was studied at twodifferent concentration doses (15 μg and 25 μg per dose) and revealedthat both concentration elicited protective antibodies in infants,children's and adults. The immunogenicity and safety of BBIL's Vi-TTconjugate vaccine's typhoid Vi capsular polysaccharide-tetanus toxoidprotein conjugate in comparison with reference vaccine (Salmonella typhiVi-polysaccharide vaccine Typbar® were evaluated.

In phase-II: A total 100 subjects were enrolled to evaluate the safetyand immunogenicity of Typhoid Vi capsular polysaccharide-TT proteinconjugate vaccine in comparison with reference Typhoid Vi capsularpolysaccharide vaccine Typbar® in healthy teenagers of 13 to 17 years ofage, children of 6-12 and 2-5 years old. The study demonstrated that thetest vaccine Vi capsular polysaccharide-tetanus toxoid conjugate (Vi-TT)as superior to the reference Typhoid Vi capsular polysaccharide vaccineTypbar® with respect to the immunogenicity and reactogenicity in all agegroups. The geometric mean of Vi IgG in terms of ELISA UNITS permilliliter (EU/ml) elevated more than four-fold raise 80%, 100% and 70%respectively when compared to the pre vaccinated sera for plain Typbar®.

The test Vaccine of Typhoid Vi Capsular Polysaccharide-tetanus toxoidconjugate Vaccine (Vi-TT) was administered 25 mcg/dose as singleinjection for age group 13-17 year teenagers and 2-6 years. Thegeometric mean of Vi IgG EU/ml elevated more than four-fold raiserespectively 100% in both the age groups when compared to the prevaccinated sera. Correspondingly the age group of 2-5 Years was injectedwith 25 μg /dose in two injections. The time interval for administrationof second injection was 6 weeks respectively. The geometric mean of ViIgG EU/ml elevated more than four-fold raise respectively 100% in thisage group when compared to the pre vaccinated sera.

Another group was designed as 15 μg /dose as two injections for the agegroup between 2-5 Years age. The time interval for administration ofsecond injection was 6 weeks respectively. The geometric mean of Vi IgGEU/ml elevated more than four-fold raise respectively 100% in the agegroup 2-5 years when compared to the pre vaccinated sera.

All test group injected with 25 μg as single injection, 25 μg as doubleinjections per dose and 15 μg as double injection per dose showed 100%seroconversion. The antibody responses to the Vi-Polysaccharide-TetanusToxoid Protein conjugate vaccine is superior to the reference nativepolysaccharide vaccine in all age groups. Hence it can be concluded thatthe test vaccine Typhoid Vi Capsular Polysaccharide Tetanus Toxoidconjugate (Vi-TT) vaccine of BBIL was immunogenic to alreadycommercially available reference vaccine Typbar® of BBIL.

In Phase-III Details of number of subjects: A total of 981 subjectsallocated to the Typhoid conjugate ViPs-TT vaccine and reference vaccineTypbar® to evaluate the immunogenicity and safety of TyphoidVi-polysaccharide-TT conjugate vaccine ViPs-TT (Typbar-TCV™) Vs. plainTyphoid Vi-polysaccharide vaccine (Typbar®, Reference vaccine). BBIL'styphoid conjugate ViPs-TT (Typbar-TCV™) vaccine, Geometric Mean Titre(GMT) and % seroconversion-4-fold was analysed between three-age groups(6 months to 2 year, greater than 2 to greater than 15 years and 15 to45 years) in comparison with plain Typhoid Vi-polysaccharide vaccine(Typbar®, Reference vaccine). The GMT in subjects in the age groupbetween 6 months to 2 years, greater than 2 to greater than 15 years and15 to 45 years in Typhoid-TT conjugate vaccine at day 42 were 1952.03EU/ml, 1555.51 EU/ml, and 812.97 EU/ml of Typhoid anti Vi IgG antibodyby ELISA respectively. The percentage of seroconversion (4-fold titrerise) in subjects in the age group between 6 months to 2 years, greaterthan 2 to greater than 15 years and 15 to 45 years in the Typhoid-TTconjugate vaccine was 98.05%, 99.17% & 92.13% respectively at day 42(FIG. 12).

TABLE 4.1 Typbar-TCV ™ phase III clinical trial data Age group 6 monthsto 2 2 years to 15 15 years to 45 Response Time period years (N = 307)years (N = 242) years (N = 90) GMT EU/ml Day 0 9.44 (8.66, 10.31)9.61(8.92, 10.35) 13.01(10.60, 15.97) (LCL, UCL) Day 42 1952.03 1555.51812.97 (1795.48, 2122.23) (1371.33, 1764.43) (637.66, 1036.46)Seroconversion Day 0 to 98.05% 99.17% 92.13% (% age) (4 fold) Day 42

In 2 to greater than 15 year age group GMT in Typhoid-TT conjugateTypbar-TCV™ vaccine and Typhoid vaccine Typbar® group on day 42 were1555.51 EU/ml and 426.63 EU/ml of Typhoid anti Vi IgG antibody by ELISArespectively (p=0.00001). The percentage of seroconversion (4-fold titrerise) on day 42 between Typbar-TCV™ vaccine and Typhoid vaccine Typbar®99.17% and 94.86% respectively (p=0.0086).

In 15- to 45-year age group GMT in Typbar-TCV™ vaccine and Typhoidvaccine Typbar® group on day 42 were 812.97 EU/ml and 376.81 EU/ml ofTyphoid anti-Vi IgG antibody by ELISA respectively (p=0.0001). Thepercentage of seroconversion (4-fold titre rise) on day 42 betweenTypbar-TCV™ vaccine and Typhoid vaccine Typbar® group 92.13% and 89.01%respectively (p=0.4737).

The superiority of Typhoid-TT (ViPs-TT) conjugate vaccine is 3.16 timeshigher than plain polysaccharide vaccine with respect to GMT postvaccination. The estimated GMT of Post to Pre titre ratio of typhoidconjugate vaccine (test) is 3.53 times higher than that of plainpolysaccharide vaccine (reference). With respect to seroconversiontyphoid conjugate vaccine is significantly superior to plainpolysaccharide vaccine at a margin of 0.016%.

Summary of phase III clinical trial data of ViPs-TT conjugate vaccine ofBBIL Typbar-TCV™ is detailed below in comparison with reference vaccineTypbar® (FIGS. 12 and 13) and as well with Peda Typh™ is provided in thebelow tables:

TABLE 4.2 Typbar-TCV ™ vs. Typbar ® Single injection of 25 μg of ViPs-TT conjugate vaccine Typbar- TCV ™ of BBIL to comprise a completevaccination schedule Plain ViPs vaccine of BBIL (single dose) Typbar ®Geometric % age Geometric % age Mean Titre at Seroconversion Mean Titreat Seroconversion Age group day 42 (EU/ml) on day 42 day 42 (EU/ml) onday 42 6 months to 24 1952.03 98.05% Not applicable Not Applicablemonths 2 years to 15 1555.51 99.17% 426.03 94.86% years 15 years to 45812.97 92.13% 376.81 89.01% years

EXAMPLE 5 TCV and Measles Interference Study

Typbar-TCV™ is a preparation of Vi-polysaccharide vaccine conjugated toTetanus Toxoid caner protein. It has been proven that children whoreceived the Vi conjugate vaccine achieved and maintained higher levelsof anti-Vi IgG serum antibodies compared to those who received the plainVi-polysaccharide vaccine. Typbar-TCV™ (ViPs-TT conjugate vaccine) isproposed in the immunization schedule to have been administered between6^(th) month to 24^(th) month, and preferably in the 9^(th) month fromchild birth. Since, Measles vaccine immunization is also done at thesame time, to combine both the vaccines and administer as a singleinjection will provide added benefits. In order to be able to do this,the interference of the two vaccines on each other's biological andchemical properties needs to be explored. In line with the aboveproposal, a study was designed to reconstitute the lyophilized Measlesvaccine with the liquid Typbar-TCV™ (ViPs-TT conjugate vaccine) andconduct O-acetyl content test (for Typbar-TCV™) and Cytopathic Effectmethod (for Measles Vaccine) at 0 hrs, 4 hrs, 8 hrs and 12 hrs followingincubation at 25° C. It was checked whether the physiochemical andbiological parameters of both the vaccines were within specifications atthe said temperature and time points. This study provided an overview ofthe laboratory findings for the reconstituted vaccine product for ashort period of time.

TABLE 5.1 Specification of Typhoid conjugate vaccine and measles vaccineTyphoid Conjugate Vaccine Measles vaccine Typbar-TCV ™ Measles vaccine(LIVE) I.P (ViPs-TT conjugate vaccine) (Freeze-dried) Single dose - 0.5mL Single dose - 0.5 mLTest Performed: O-Acetyl content by Hestrin's Method

Vi polysaccharide is a linear homopolymer composed of(1-4)-20acetamido-2-deoxy-α-D-galacturonic acid that is O-acetylated atcarbon-3. The O-acetyl content of the purified Vi-polysaccharide isimportant for the immunogenicity of Vi and it can be measured by usingHestrin's method.

TABLE 5.2 O-acetyl content by Hestrin method S. No. Sample Detail 0 Hour4^(TH) Hour 8^(TH) Hour 1. Typbar-TCV ™ (ViPs-TT 0.098 μmoles/dose 0.098μmoles/dose 0.098 μmoles/dose conjugate vaccine) at the start of timepoint 2-8° C. 2. Typbar-TCV ™ (ViPs-TT 0.100 μmoles/dose 0.100μmoles/dose 0.096 μmoles/dose conjugate vaccine) kept at 25° C. 3.Measles Vaccine 0.151 μmoles/dose 0.086 μmoles/dose 0.058 μmoles/dosereconstituted with Typbar- TCV ™ (ViPs-TT conjugate vaccine) and kept at25° C. Specification 0.064-0.106 μmoles/dose 0.064-0.106 μmoles/dose0.064-0.106 μmoles/dose

Results: The Measles vaccine reconstituted with Typbar-TCV™ (ViPs-TTconjugate vaccine) was incubated at 25° C. was analyzed for O-acetylcontent by Hestrin's method. As controls, the Typbar-TCV™ (ViPs-TTconjugate vaccine) kept at 2-8° C. and Typbar-TCV™ (ViPs-TT conjugatevaccine) at the start of time point 25° C. were also analyzedsimultaneously. As expected, the O-acetyl content of the control samplesat 2-8° C. and 25° C. were close to the initial value. The O-acetylcontent of the combination vaccine (Measles+TCV) was higher than theacceptance criteria at 0 hrs (0.151 μmoles/dose). It decreased with timeat 4 hrs and 8 hrs (0.086 and 0.058 μmoles/dose) which were withinacceptance criteria, but different when compared to the Typbar-TCV™ onlyvalues at 2-8° C. and 25° C.

Test Performed: Potency Test by Cytopathic Effect (CPE) Method

Measles Vaccine is a live attenuated vaccine. To titrate the measlesvaccine logarithmic dilution was prepared, each logarithmic dilutioninoculated in to vero cell line with 8 replicates and incubated for 7-8days and checked for the presence or absence of Cytopathic Effect. Virustitre is calculated by Spearman Karber formula. Results are as below:

TABLE 5.3 Potency test by Cytopathic Method Results (log10 CCID50/0.5mL) of Measles Interference Study with TCV S. 0 4 8 12 No. Sample DetailHour Hour Hour Hours 1 Measles Vaccine reconstituted with 3.50 3.40 3.50its diluent at the start of each time point 2 Measles Vaccinereconstituted with 3.50 3.45 3.50 3.40 its diluent and kept at 25° C. 3Measles Vaccine reconstituted with 3.30 3.15 3.00 2.80 Typbar-TCV ™(ViPs-TT conjugate vaccine) and kept at 25° C. Specification NLT 3.00Results: From the results, it is observed that Measles vaccine whenreconstituted with its diluent, found stable for 12 hours and whenreconstituted with the Typbar-TCV™ (ViPs-TT conjugate vaccine) is stablefor 4 hours and fell between 4 and 8 hours.

Other embodiments and uses of the invention will be apparent to thoseskilled in the art from consideration of the specification and practiceof the invention disclosed herein. All references cited herein,including all publications, U.S. and foreign patents and patentapplications, are specifically and entirely incorporated by reference.It is intended that the specification and examples be consideredexemplary only with the true scope and spirit of the invention indicatedby the following claims. Furthermore, the term “comprising of” includesthe terms “consisting of” and “consisting essentially of.”

We claim:
 1. A method of manufacture of a ViP-carrier protein conjugatecomprising: purifying capsular Vi-polysaccharide antigens (VIPs) frombacteria; treating the purified VIPs with sodium bicarbonate formingpartially de-O-acetylated ViPs; treating the partially de-O-acetylatedViPs with a carbodiimide cross-linking agent; linking the carbodiimidetreated partially de-O-acetylated ViPs with a linker molecule; isolatingthe linked partially de-O-acetylated ViPs; and contacting the linkedpartially de-O-acetylated ViPs with a carrier protein in presence ofEDAC to form the ViP-carrier protein conjugate.
 2. The method of claim1, wherein the antigen comprises a native or recombinant antigen.
 3. Themethod of claim 1, wherein the bacteria comprises Salmonella.
 4. Themethod of claim 1, wherein the bacterial comprises Salmonella entericaserovar typhi or Salmonella enterica serovar paratyphi.
 5. The method ofclaim 1, wherein the purified VIPs have an approximate molecular weightof 250-300 kDa.
 6. The method of claim 1, wherein the purified VIPs areat a concentration of about 5-7.5 mg/ml.
 7. The method of claim 1,wherein the treating the purified VIPs with sodium bicarbonate comprisesa microwave treatment and/or high pressure homogenization.
 8. The methodof claim 1, wherein the carbodiimide cross-linking agent comprises1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDAC).
 9. The method ofclaim 1, wherein the linker molecule comprises adipic acid dihydrazide.10. The method of claim 1, wherein the carrier protein comprises abacterial or viral protein.
 11. The method of claim 1, wherein thecarrier protein comprises diphtheria toxoid, CRM197, tetanus toxoid,Pseudomonas exoprotein A, Pseudomonas aeruginosa toxoid, Bordetellapertusis toxoid, Clostridium perfringens toxoid, Escherichia coliheat-labile toxin B subunit, Flagellin Fli C, or Horseshoe crabHaemocyanin.
 12. The method of claim 1, wherein the a ViP-carrierprotein conjugate is purified and/or sterilized.
 13. The method of claim1, wherein the VIP-carrier protein has an approximate molecular weightof less than 1000 kDa.
 14. The method of claim 1, wherein theVIP-carrier protein has an approximate molecular weight of about 250-300kDa.
 15. The method of claim 1, wherein the percent of freepolysaccharide in the ViP-carrier protein conjugate is from about 3% to6%.
 16. The method of claim 1, wherein the ViP content/protein contentof the ViP-carrier protein conjugate is about 1.1 to 1.2.
 17. The methodof claim 1, wherein the yield of conjugate ViPs-carrier protein moleculeranges from 70% to 80%.
 18. The method of claim 1, further comprisingcontacting the ViP-carrier protein conjugate to a preservative.
 19. Themethod of claim 19, wherein the preservative comprises 2-phenoxy ethanolwhich is free of mercury chloride and/or thiomersal.
 20. A method ofmanufacture of a ViP-carrier protein conjugate comprising: treatingcapsular Vi-polysaccharide antigens (VIPs) of Salmonella typhi withsodium bicarbonate forming partially de-O-acetylated ViPs; treating thepartially de-O-acetylated ViPs with a carbodiimide cross-linking agent,wherein the cross-linking agent comprises1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDAC); linking thecarbodiimide treated partially de-O-acetylated ViPs with a linkermolecule; isolating the linked partially de-O-acetylated ViPs; andcontacting the linked partially de-O-acetylated ViPs with a carrierprotein in presence of EDAC to form the ViP-carrier protein conjugate.21. The method of claim 20, wherein the carrier protein comprisesdiphtheria toxoid, CRM197, tetanus toxoid, Pseudomonas exoprotein A,Pseudomonas aeruginosa toxoid, Bordetella pertusis toxoid, Clostridiumperfringens toxoid, Escherichia coli heat-labile toxin B subunit,Flagellin Fli C, or Horseshoe crab Haemocyanin.
 22. The method of claim20, wherein the carrier protein comprises diphtheria toxoid, CRM197, ortetanus toxoid.
 23. The method of claim 20, wherein the linker moleculecomprises adipic acid dihydrazide.
 24. The method of claim 20, whereinthe percent of free polysaccharide in the ViP-carrier protein conjugateis from about 3% to 6%.
 25. The method of claim 20, wherein the ViPcontent/protein content of the ViP-carrier protein conjugate is about1.1 to 1.2.
 26. A method of manufacture of an immunogenic compositioncontaining a ViP-carrier protein conjugate comprising: treatingpurifying capsular Vi-polysaccharide antigens (VIPs) of Salmonella typhiwith sodium bicarbonate forming partially de-O-acetylated ViPs with amolecular weight of from 250-300 kDa; treating the partiallyde-O-acetylated ViPs with a carbodiimide cross-linking agent; linkingthe carbodiimide treated partially de-O-acetylated ViPs with a linkermolecule, wherein the linker molecule comprises adipic acid dihydrazide;isolating the linked partially de-O-acetylated ViPs from free linkermolecules; contacting the linked partially de-O-acetylated ViPs with acarrier protein in presence of EDAC to form the ViP-carrier proteinconjugate; and contacting the ViP-carrier protein conjugate with apreservative, wherein the preservative comprises 2-phenoxy ethanol whichis free of mercury chloride and/or thiomersal.
 27. A method ofmanufacture of a ViP-carrier protein conjugate comprising: treatingcapsular Vi-polysaccharide antigens (VIPs) of Salmonella typhi withsodium bicarbonate forming partially de-O-acetylated ViPs with amolecular weight of from 250-300 kDa; and conjugating the partiallyde-O-acetylated ViPs with a carrier protein to form the ViP-carrierprotein conjugate.
 28. The method of claim 27, wherein treating furthercomprises a microwave treatment and/or high pressure homogenization. 29.The method of claim 27, wherein the carrier protein comprises abacterial or viral protein.
 30. The method of claim 27, wherein thecarrier protein comprises diphtheria toxoid, CRM197, tetanus toxoid,Pseudomonas exoprotein A, Pseudomonas aeruginosa toxoid, Bordetellapertusis toxoid, Clostridium perfringens toxoid, Escherichia coliheat-labile toxin B subunit, Flagellin Fli C, or Horseshoe crabHaemocyanin.
 31. The method of claim 27, wherein the ViP-carrier proteinconjugate is purified and/or sterilized.
 32. The method of claim 27,wherein the ViP-carrier protein conjugate is mixed with a preservative.33. The method of claim 32, wherein the preservative comprises 2-phenoxyethanol which is free of mercury chloride and/or thiomersal.